Familial insulin-resistant diabetes secondary to an affinity defect of the insulin receptor

We describe three siblings with a mild diabetes mellitus in combination with acanthosis nigricans and multiple minor physical abnormalities. Fasting plasma insulin was elevated up to 100-fold as compared with normal values, and the diabetes was classified as insulin resistant. Insulin-binding studies on erythrocytes, monocytes, and cultured fibroblasts disclosed an abnormally reduced binding capacity, as compared with that of healthy controls, which was most prominent at low concentrations of insulin. Scatchard analysis on erythrocytes of the three patients revealed a normal number of total insulin-binding sites per cell, but a complete lack of insulin binding to the high-affinity receptor component. The findings are consistent with the assumption of two genetically distinct types of insulin receptors.     

Glucagon-like peptide-1 improves insulin and proinsulin binding on RINm5F cells and human monocytes

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.     

Insulin activation of NADH ferricyanide reductase in human erythrocytes is mediated by the insulin receptor tyrosine kinase: a comparative study in normal and diabetic states

Summary

We have previously reported that NADH ferricyanide reductase in human erythrocytes is stimulated by insulin. Hormone-stimulated activities are attenuated in the presence of glycolytic inhibitors like vanadate, indicating the involvement of glycolysis in the mechanism by which insulin stimulates ferricyanide reduction. Activation of erythrocyte metabolism in response to insulin could be a result of hormone binding to its receptor, inducing phosphorylation of band 3 (at a site for reversible association of glycolytic enzymes) and/or other membrane proteins like the Na⁺/H⁺ antiport. Activation of the antiporter protein by insulin can stimulate glycolysis by an increase in intracellular pH, an effect which is prevented by amiloride. Evidence for a role for tyrosine phosphorylation in triggering the reductase activation came from studies with protein kinase inhibitors. Genistein, sphingosine and acridine orange have been shown to prevent insulin-stimulated ferricyanide reduction, implicating tyrosine phosphorylation as an important signal for activation of the enzyme by insulin. To evaluate activation of the enzyme by insulin stimulated phosphorylation, a comparative study was done using erythrocytes from healthy and diabetic humans. We measured ferricyanide reductase activities in basal and insulin stimulated states. Basal activities were lower in diabetics than in normal humans. Nevertheless, hormone stimulated activities were similar, despite earlier reports of decreased receptor phosphorylation of exogenous substrates in type 2 diabetics. These observations, together with previous ones, suggest that insulin-receptor kinase interaction may mediate the action of insulin on human erythrocytes by phosphorylation of cellular proteins like band 3 and/or the Na⁺/H⁺ antiport.     

Impaired 6-phosphofructokinase activity in mononuclear leukocytes from patients with type II diabetes mellitus

Seven cytoplasmic enzyme activities were measured in extracts of mononuclear leukocytes (lymphocytes plus monocytes) obtained from 19 type II diabetic humans and 10 healthy control subjects. 6-Phosphofructokinase activity was significantly decreased in cell extracts from diabetics, while other enzyme activities were similar in diabetics and controls. Since the effects of starvation on enzyme activities are sometimes similar to the effects of diabetes, the studies were repeated in 5 control subjects after a 2-day fast. This short period of starvation did not mimic the effect of diabetes on 6-phosphofructokinase activity. The decreased enzyme activity was not correlated with percent specific insulin binding to monocytes in the same cell preparations nor to clinical variables such as obesity or the broad range of fasting plasma glucose values encountered among the diabetics. We conclude that 6-phosphofructokinase activity in mononuclear leukocytes, as in other tissues, may be a marker for a postreceptor lesion associated with the insulin resistance found in type II diabetes mellitus.     

Characterization of the insulin resistance in liver cirrhosis: a comparison with non-insulin dependent diabetes mellitus.

To characterize the mechanisms of insulin resistance in liver cirrhosis (LC), we estimated the peripheral tissue sensitivity and responsiveness to insulin using the euglycemic clamp technique and determined the insulin binding to erythrocytes in patients with compensated LC as well as in patients with non-insulin dependent diabetes mellitus (NIDDM). The insulin dose-response curves of the glucose metabolic clearance rates (MCR) were shifted to the right and downward both in patients with LC and NIDDM, indicating a reduced sensitivity and responsiveness to insulin. In the cirrhotics, MCR at the maximally effective insulin level, an index of insulin responsiveness, was correlated with fasting insulin levels (r = -0.57, P < 0.01) and sigma BG in 75 gOGTT (r = -0.43, P < 0.05), but no correlations were found between them and the diabetics. Although specific insulin bindings to erythrocytes were significantly lower in patients both with LC and NIDDM, Scatchard analysis revealed a significant decrease in the number of insulin receptors in the cirrhotics, and a decrease in the empty-site affinity in the diabetics. These findings suggest that insulin resistance in LC consists of a combination of binding and postbinding defects. The latter defect may be caused by basal hyperinsulinemia and contribute to the development of glucose intolerance. Although binding and postbinding abnormalities are also found in NIDDM, the mechanisms of insulin resistance in LC and NIDDM may be different.     

Effect of tetrahydrocurcumin on insulin receptor status in type 2 diabetic rats: studies on insulin binding to erythrocytes

Curcumin is the most active component of turmeric. It is believed that curcumin is a potent antioxidant and anti-inflammatory agent. Tetrahydrocurcumin (THC) is one of the major metabolites of curcumin, and exhibits many of the same physiological and pharmacological activities as curcumin and, in some systems, may exert greater antioxidant activity than curcumin. Using circulating erythrocytes as the cellular mode, the insulin-binding effect of THC and curcumin was investigated. Streptozotocin (STZ)-nicotinamide-induced male Wistar rats were used as the experimental models. THC (80 mg/kg body weight) was administered orally for 45 days. The effect of THC on blood glucose, plasma insulin and insulin binding to its receptor on the cell membrane of erythrocytes were studied. Mean specific binding of insulin was significantly lowered in diabetic rats with a decrease in plasma insulin. This was due to a significant decrease in mean insulin receptors. Erythrocytes from diabetic rats showed a decreased ability for insulin-receptor binding when compared with THC-treated diabetic rats. Scatchard analysis demonstrated that the decrease in insulin binding was accounted for by a decrease in insulin receptor sites per cell, with erythrocytes of diabetic rats having less insulin receptor sites per cell than THC-treated rats. High affinity (K d1), low affinity (K d2) and kinetic analyses revealed an increase in the average receptor affinity of erythrocytes from THC-treated rats compared with those of diabetic rats. These results suggest that acute alteration of the insulin receptor on the membranes of erythrocytes occurred in diabetic rats. Treatment with THC significantly improved specific insulin binding to the receptors, with receptor numbers and affinity binding reaching near-normal levels. Our study suggests the mechanism by which THC increases the number of total cellular insulin binding sites resulting in a significant increase in plasma insulin. The effect of THC is more prominent than that of curcumin.     

Erythrocyte insulin binding in normal infants, children and adults

To establish normal insulin binding criteria, we studied the binding of insulin to erythrocytes from normal subjects of different ages. Insulin binding to cord erythrocytes and to erythrocytes from infants aged 2-7 days was significantly higher at tracer and physiological insulin concentrations than was binding to cells from children aged 1-15 years and adults. In infants aged 1-12 months the maximum insulin binding to erythrocytes was significantly higher than that to erythrocytes from children, and in addition, it correlated negatively with age. An increase in receptor concentration was found in cord erythrocytes whereas an increased receptor affinity for insulin was found in erythrocytes from infants. Insulin binding characteristics in erythrocytes from prepubertal and pubertal children were basically similar to those in women. Erythrocytes from men bound significantly higher amounts of insulin than did those from women. This difference was associated with changes in receptor affinity for insulin. There was no correlation between the insulin binding characteristics and the circulating concentration of insulin or C-peptide. The increased erythrocyte insulin binding at birth persisted over the neonatal period. There was an overall negative correlation between the maximum insulin binding and age in the subjects studied, but the major decrease in erythrocytes insulin binding occurred during the first year of life past the neonatal period. These observations stress the importance of using age-matched controls in studies on erythrocyte insulin binding in disease states.     

The erythrocyte insulin receptor response to insulin induced hypoglycaemia

The response of the erythrocyte insulin receptor to a prolonged intravenous infusion of insulin has been measured in normal individuals during hypoglycaemia and when hypoglycaemia was prevented by the concurrent infusion of glucose. When euglycaemia was maintained, mean (+/- S.D.) specific insulin binding following the 5 hour insulin infusion was unchanged (6.9 +/- 2.1 to 6.65 +/- 2.2% bound per 2.25 X 10(9) erythrocytes). In the presence of mild hypoglycaemia, mean (+/- SD) specific insulin binding rose from 6.6 +/- 2.3 to 7.6 +/- 2.5% bound per 2.25 X 10(9) erythrocytes (P less than 0.01), after 5 hours. This increase was due to increased receptor affinity. It was not correlated with the increase in the concentration of any individual counter-regulatory hormone. Initial insulin receptor binding correlated strongly with the subsequent decline in plasma glucose concentration (r = 0.9527; P less than 0.01). Thus, acute hyperinsulinaemia, when associated with hypoglycaemia, does not result in downregulation of insulin receptors on erythrocytes but rather results in increased receptor binding. Consequently, the insulin receptor may not play an active role in protecting the individual against acute hypoglycaemia.     

Effects of acute exercise on insulin binding to erythrocytes in normal men

Ten normal men were subjected to acute mild and moderate exercise tests, and the insulin binding to erythrocytes was determined before and immediately after exercise and also 10, 30 and 60 minutes after exercise in each test. The insulin binding significantly increased immediately after acute moderate exercise and did not increase immediately after acute mild exercise. In contrast, it decreased below the basal level at 30 and 60 minutes in each test. The changes in insulin receptor binding were due mainly to an alteration in insulin receptor affinity rather than a change in receptor number. These results suggest that isolated erythrocytes may be of some value for study of the effect of physical exercise on the insulin receptor.     

Time dependent effects of dexamethasone on serum insulin level and insulin receptors in rat liver and erythrocytes

The effects of glucocorticoid excess on regulation of insulin receptors were investigated in dexamethasone-treated rats. Glucocorticoid excess was produced by administration of dexamethasone (0.5 mg/100 g b.w.) 30 min, 4, 12, 18, 24, 42 or 70 h before experiments. This treatment caused time-dependent changes of glucose and insulin concentration in blood, as well as in amounts of specific insulin binding and insulin receptors of liver cells and erythrocytes. The time intervals in which dexamethasone produced the increase in insulin concentration were accompanied with decrease in insulin binding to receptors in membranes of liver cells, while significant changes in insulin binding to receptors of erythrocytes were not observed under the same experimental conditions. The effect is maximal 18 and 42 h after dexamethasone treatment that increase insulin blood level by about 85% and 60%, respectively. Receptor analysis revealed that changes in specific binding of insulin could be due to significant changes in amount of binding sites on cell surface rather than to mild alteration in receptor affinity. These findings suggest that besides the changes in insulin level, the alterations in insulin receptor number and affinity may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess.     

Defect in insulin receptor phosphorylation in erythrocytes and fibroblasts associated with severe insulin resistance

The insulin receptor is a tyrosine-specific protein kinase. Upon binding of the hormone, the kinase is activated resulting in autophosphorylation of the receptor. This kinase activity has been postulated to be an early step in the transmembrane signaling produced by insulin. To evaluate the physiologic relevance of receptor phosphorylation, we have studied insulin binding and autophosphorylation properties using cells from an individual with a variant of the Type A syndrome of severe insulin resistance and acanthosis nigricans. Erythrocytes and cultured fibroblasts from this individual exhibited normal or near normal 125I-insulin binding. Receptors extracted from erythrocytes with Triton X-100 also exhibited normal 125I-insulin binding and competition curves. Despite this, receptors extracted from both erythrocytes and fibroblasts showed a 50% decrease in insulin-stimulated autophosphorylation. Partially purified receptors from the patient's fibroblasts also exhibited a 40% decrease in their ability to phosphorylate exogenous substrates. These data suggest that the insulin resistance in this syndrome is due to a genetic abnormality which impairs insulin receptor phosphorylation and kinase activity and further support the possible role of receptor phosphorylation and kinase activity in insulin action.     

Whole body hyperthermia of rats decreases insulin binding to erythrocytes

125I-insulin binding to rat erythrocytes was studied to investigate the effect of whole body hyperthermia on the insulin receptor. Heat treatment of rats at 42 degrees C for 15 min caused a significant decrease (48.7% of control) in 125I-insulin binding to rat erythrocytes. Scatchard analysis showed that the decreased binding resulted from a decrease in the number of the insulin receptors rather than from a decrease in receptor affinity. The decreased receptor number for insulin showed no evidence of recovery, 2 h and 8 h after the hyperthermia. Plasma insulin levels remained lower than the control, up to 8 h after the hyperthermia, whereas plasma glucose, which decreased immediately after the hyperthermia, increased higher than the control, 8 h after the hyperthermia. The low plasma insulin level and decreased number of insulin receptor are believed to be possible factors for the elevation of plasma glucose.     

Defective mononuclear phagocyte function in systemic lupus erythematosus: dissociation of Fc receptor-ligand binding and internalization

Fc receptor-mediated mononuclear phagocyte system (MPS) clearance is impaired in systemic lupus erythematosus (SLE) and may contribute to the pathogenesis of the immune complex disease. To investigate the basis of MPS dysfunction, we have examined concurrent in vivo and in vitro Fc receptor function in 22 patients with SLE and 23 disease-free adults. Blood monocyte Fc receptor binding was increased rather than decreased as predicted by the saturation hypothesis of MPS blockade. Rosette formation of IgG-sensitized bovine erythrocytes (EA) with monocytes demonstrated increased Fc receptor-ligand binding in SLE (percent rosettes: 40 +/- 12 vs 27 +/- 8, p less than 0.001). Scatchard analysis of the binding of radiolabeled IgG oligomers to SLE monocytes indicated a mean receptor number 30% higher than control, although this did not reach statistical significance. Despite enhanced Fc receptor-ligand (EA) binding, Fc-mediated phagocytosis of EA was decreased in SLE (1.7 +/- 0.7 erythrocytes/monocytes/hour vs 2.6 +/- 1.0, p less than 0.004). This decrease in phagocytosis by blood monocytes from SLE patients was significantly greater than that attributable to the predominance in SLE of individuals with certain HLA B cell alloantigens and intrinsically lower phagocytic rates (p less than 0.05 for all groups). This decrease therefore represents a disease-acquired characteristic. Furthermore, the phagocytic rate of the four SLE patients with marked prolongation in MPS clearance was significantly lower than that of the eight patients with near normal clearance values (p less than 0.01). Saturation of Fc receptors by immune complexes does not explain impaired immune clearance in SLE. Our results indicate that despite increased binding of the EA ligand, Fc receptor-mediated phagocytosis is markedly impaired in SLE monocytes. This impairment cannot be explained on the basis of HLA-related differences in phagocytosis among lupus patients. The defect in phagocytosis of EA is most profound in those patients with the most significantly impaired MPS clearance. Thus, the dissociation of receptor-ligand binding and receptor-mediated internalization may contribute significantly to the in vivo clearance defect in SLE.     

Insulin receptor processing as a function of erythrocyte age. A kinetic model for down-regulation

The effects of cell aging on insulin binding and on insulin receptor processing in human erythrocytes were studied. Erythrocytes were found to exponentially lose equal proportions of both high and low affinity receptors as a function of age. The affinities of remaining surface receptors did not change significantly. The maximum extent of insulin receptor down-regulation that could be induced decreased linearly with age over the range studied. Together with dose-response and time course studies, these age-related changes in insulin binding and receptor down-regulation were used to develop a kinetic model in which receptor internalization is a function of surface receptor concentration. The ability of the model to predict the behavior of a heterogeneous population suggests that changes in receptor processing with age may be attributed to changes in the surface receptor concentration.     

Absence of covalent binding by insulin to erythrocyte and reticulocyte insulin receptors

We investigated whether insulin forms covalent bonds with its receptors on erythrocytes and reticulocytes, as it does in adipocytes (1). Of the [125I]-insulin specifically bound at 37 degrees C to human and rat erythrocytes and rat reticulocytes, only 1.5-2.3% was non-dissociable on extensive washing. When ghosts prepared from the washed cells were solubilized in Triton X-100, only 0.6-1.5% of the specifically bound radioactivity appeared in the void volume of a Sephadex G-50 column. Moreover in contrast to adipocytes, this high molecular weight radioactivity was not immunoprecipitable by antibodies to the insulin receptor and was dissociated during chromatography in sodium dodecyl sulphate. Thus we have been unable to demonstrate the formation of covalent bonds between insulin and its receptors on erythrocytes and reticulocytes. This finding is consistent with the hypothesis that covalent binding of insulin is a necessary receptor modification for insulin's metabolic effects.     

Altered phagocytosis by monocytes from HLA-DR2 and DR3-positive healthy adults is Fc gamma receptor specific

Fc gamma receptor-dependent mononuclear phagocyte system (MPS) clearance of opsonized erythrocytes is prolonged in healthy adults with the class II alloantigens HLA-DR2, DR3, or DQw1, despite normal receptor-specific Fc ligand binding by monocytes in these groups. To investigate the basis for the MPS dysfunction, we determined the phagocytic capacity of blood monocytes from 66 disease-free adults and analyzed the data according to the HLA type of the subjects. The data demonstrate decreased phagocytosis of IgG-sensitized erythrocytes (EA) by monocytes from HLA-DR2, DR3, or DQw1-positive subjects compared with normals without these B cell alloantigens (2.87 +/- 0.83 erythrocytes/monocyte vs 3.87 +/- 1.05, p less than 0.004; 3.01 +/- 0.94 vs 3.87 +/- 1.05, p less than 0.02; 3.18 +/- 0.89 vs 3.87 +/- 1.05, p less than 0.02, respectively). Because HLA-DR2 and DQw1 are in linkage disequilibrium, we compared EA phagocytosis in subjects with DQw1 alone to normals without HLA-DR2, DR3, or DQw1. Among subjects positive only for DQw1, no significant decrease in phagocytosis could be seen (3.46 +/- 0.95 vs 3.87 +/- 1.05, p = NS). To determine whether these differences represented an Fc receptor-specific dysfunction or a more generalized decrease in phagocytic activity, we compared the quantitative phagocytosis of EA with that of neuraminidase-treated erythrocytes (EN), which is Fc receptor independent and beta-glucan receptor mediated. No segregation of phagocytic capacity for EN by HLA class II phenotypes could be demonstrated (DR2, 2.68 +/- 1.30 erythrocytes/monocyte; DR3, 2.95 +/- 1.30; DQw1, 2.84 +/- 1.15; others, 3.06 +/- 1.14). Our data indicate that the decrease in phagocytosis by blood monocytes from normal individuals with HLA-DR2 or DR3, the class II alloantigens associated with systemic lupus erythematosus and other autoimmune diseases, is specific for the Fc receptor-mediated process. This alteration of Fc receptor function among immunogenetically defined individuals may contribute to their predisposition to autoimmune disease. These differences may also apply to other Fc receptor-initiated cellular functions.     

Effects of crush syndrome on insulin receptor interactions in cells

Comparative study of binding insulin receptor mononuclears, erythrocytes and hepatocytes of crush-syndrome revealed great difference in these parameters. In the early period of crush-syndrome (stress period insulin insufficiency hyperglycemia, insulin resistance, decrease in the activity of insulin binding in the mononuclears and increase in the activity of erythrocytes. By the 6 hour crush-syndrome (toxemic period) hyperinsulinemia, hyperglycemia, are observed as well as prolongation of the decrease in the activity of insulin binding of mononuclears.     

Insulin receptors in rabbit erythrocyte

The existence of insulin receptors in rabbit erythrocytes was studied by evaluating the specific binding of 125I-insulin to erythrocyte membranes. The binding of 125I-insulin was pH, time and temperature dependent. Maximal binding was achieved by incubation for 20 hr at 0 degrees C. The optimum pH was 7.4. Treatment with cations and enzymes enhanced the specific binding except for with trypsin, the treatment which greatly reduced the binding. Unlabeled insulin over a wide range of concentrations competitively inhibited the binding of 125I-insulin, while the binding was little affected by structurally unrelated hormones. Scatchard plot was represented as a concave curve. Binding sites of relatively high affinity (K1 = 0.9 X 10(9) M-1) and low capacity (8.0 X 10(13)/g protein) could be distinguished from those of lower affinity (K2 = 0.8 X 10(7) M-1) and higher capacity (1.8 X 10(15)/g protein). Hill's analysis and dissociation of 125I-insulin from membranes demonstrated the characteristics of negative cooperation between receptor sites. Both incorporation of H3(32)PO4 to erythrocyte membranes and uptake of 45Ca were significantly reduced by the addition of unlabeled insulin. Unlabeled insulin produced no effect on uptake of 45Ca into trypsin-treated erythrocytes. On the basis of these results, it was suggested that rabbit erythrocytes might possess biologically significant insulin receptors located on the cell membranes.     

The insulin receptor of the turkey erythrocyte. Characterization of the membrane-bound receptor.

The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.